Cutting Edge: Lectin-Like Transcript-1 Is a Ligand for the Inhibitory Human NKR-P1A Receptor This information is current as David B. Rosen, Jayaram Bettadapura, Mohammed of September 27, 2021. Alsharifi, Porunelloor A. Mathew, Hilary S. Warren and Lewis L. Lanier J Immunol 2005; 175:7796-7799; ; doi: 10.4049/jimmunol.175.12.7796 http://www.jimmunol.org/content/175/12/7796 Downloaded from References This article cites 27 articles, 15 of which you can access for free at: http://www.jimmunol.org/content/175/12/7796.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 27, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. THE JOURNAL OF IMMUNOLOGY CUTTING EDGE Cutting Edge: Lectin-Like Transcript-1 Is a Ligand for the Inhibitory Human NKR-P1A Receptor1 David B. Rosen,* Jayaram Bettadapura,† Mohammed Alsharifi,† Porunelloor A. Mathew,‡ Hilary S. Warren,2† and Lewis L. Lanier2,3* Increasingly, roles are emerging for C-type lectin receptors mAb inhibits human NK cell-mediated cytotoxicity against ϩ in immune regulation. One receptor whose function has FcR target cells (4, 5). remained largely enigmatic is human NKR-P1A Recently, ligands for two mouse Klrb1 family members have (CD161), present on NK cells and subsets of T cells. In this been identified (10, 11). The activating Nkr-p1f receptor rec- study, we demonstrate that the lectin-like transcript-1 ognizes Clr-g (encoded by Clec2i) (10), and the inhibitory Nkr- (LLT1) is a physiologic ligand for NKR-P1A. LLT1-con- p1d recognizes Clr-b (encoded by Clec2d) (11). Another name Downloaded from -؉ for Clr-b is osteoclast inhibitory lectin, because it was also iden taining liposomes bind to NKR-P1A cells, and binding tified as an osteoblast-derived glycoprotein, which inhibits in is inhibited by anti-NKR-P1A mAb. Additionally, LLT1 ␨ vitro osteoclastogenesis (12, 13). Although mice have multiple activates NFAT-GFP reporter cells expressing a CD3 - Clr family genes, only one ortholog, CLEC2D (also named lec- NKR-P1A chimeric receptor; reciprocally, reporter cells tin-like transcript-1 (LLT1)4), exists in humans. Like mouse with a CD3␨-LLT1 chimeric receptor are stimulated by Clr-b, human LLT1 blocks osteoclast differentiation (12, 13). http://www.jimmunol.org/ NKR-P1A. Moreover, LLT1 on target cells can inhibit Mouse and human Clec2 gene products are type II proteins of NK cytotoxicity via interactions with NKR-P1A. The the C-type lectin superfamily. Journal of Immunology, 2005, 176: 7796–7799. The ability of mouse Nkrp1 family receptors to recognize Clr ligands prompted the question of whether this interaction is conserved in humans. In this study, we have examined whether odents have several Klrb1 (also named Nkrp1) genes LLT1 serves as a ligand of human NKR-P1A and have de- encoding either activating or inhibitory NK receptors scribed the functional consequences of this novel receptor-li- of the C-type lectin superfamily, including Nkr-p1c, R gand interaction. by guest on September 27, 2021 the NK1.1 Ag defining mouse NK cells (1). By contrast, only a single, nonpolymorphic gene in the Nkrp1 family, KLRB1, ex- ists in humans (2). KLRB1 encodes a type II disulfide-linked Materials and Methods LLT1 liposomes homodimer, named CD161 or NKR-P1A, which is expressed on most NK cells. Human NKR-P1A is on a subset of periph- A cDNA encoding the extracellular domain of LLT1 with a N-terminal 6-His ϩ ϩ tag was cloned into a pFASTBac vector, modified to encode a signal sequence eral T cells, including CD4 and CD8 T cells (mostly effec- ϩ for ecdysteroid UDP glucosyltransferase, and expressed in the Bac-to-Bac sys- tor/memory phenotype), invariant NKT cells, and ␥␦-TCR T tem (Invitrogen). Recombinant protein was purified by Ni-NTA affinity chro- ϩ cells, and on a subset of CD3 thymocytes (2). matography. The preparation of LLT1-fluorescent liposome complexes and the procedure for binding to cells was described previously (14–16). To ensure NKR-P1A is expressed on immature human NK cells, before binding was specific, cells were incubated with an anti-CD161 mAb or an iso- acquisition of CD16 or CD56 (3), and its expression is up-reg- type-matched control Ig (10 ␮g/ml) before incubating with the liposome- ϩ ulated on mature NK cells by IL-12 (4, 5). NKR-P1A on CD4 rLLT1 complexes. ϩ T cells and ␥␦-TCR T cells has been implicated in transen- Reporter cell assays dothelial migration (6, 7). Additionally, anti-NKR-P1A mAbs costimulate the anti-CD3 mAb-induced proliferation of An NFAT-GFP reporter construct was stably transduced into 2B4 T cell hy- bridoma cells (provided by H. Arase, Osaka University, Osaka, Japan). NFAT- CD1d-specific NK T cells (8) and induce proliferation of im- GFP reporter cells were transduced with CD3␨-LLT1 or CD3␨-NKR-P1A chi- mature thymocytes (9). Cross-linking with an anti-NKR-P1A meric receptors and analyzed as described (17, 18). Briefly, reporter and *Department of Microbiology and Immunology, The Cancer Research Institute, and Bio- Fellow of the National Health and Medical Research Council of Australia, and a Visiting medical Sciences Graduate Program, University of California, San Francisco, CA 94143; Fellow of the Australian National University Medical School and The Canberra Hospital. †Division of Immunology and Genetics, The John Curtin School of Medical Research, L.L.L. is an American Cancer Society Research Professor. D.B.R. is supported by a The Australian National University, Canberra City, Australian Capital Territory, Austra- Genentech Graduate Student Fellowship. lia; and ‡Department of Molecular Biology and Immunology and Institute for Cancer 2 H.S.W. and L.L.L. contributed equally to this work. Research, University of North Texas Health Science Center, Fort Worth, TX 76107 3 Address correspondence and reprint requests to Dr. Lewis L. Lanier, Department of Mi- Received for publication September 27, 2005. Accepted for publication October 28, 2005. crobiology and Immunology, University of California San Francisco, 513 Parnassus Ave- The costs of publication of this article were defrayed in part by the payment of page charges. nue, Box 0414, San Francisco, CA 94143-0414. E-mail address: [email protected] This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. 4 Abbreviations used in this paper: LLT1, lectin-like transcript-1; MICA, MHC class I Section 1734 solely to indicate this fact. chain-related A. 1 This work was supported by National Institutes of Health Grant AI068129 and National Health and Medical Research Council Project Grant 151304. H.S.W. is a Senior Research Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 The Journal of Immunology 7797 stimulator cells were cocultured at a 1:4 ratio for 16 h and then analyzed for GFP expression by flow cytometry. Constructs and transductions cDNAs for human NKp80 (provided by J. P. Houchins, R&D Systems, Min- neapolis, MN), human CD69, LLT1 with a C-terminal Flag-epitope, human NKR-P1A, and the reporter constructs were subcloned into pMXs-puro vectors (provided by T. Kitamura, University of Tokyo, Tokyo, Japan) (19). Chimeric receptors were generated by fusing the extracellular and transmembrane do- mains of either LLT1 or NKR-P1A to the intracellular domain of human CD3␨. Plasmid constructs were transfected into Phoenix packaging cells (20) to produce retroviruses (18, 21). Infected cells were selected in medium contain- ing 1 ␮g/ml puromycin. Antibodies Mouse anti-human NKR-P1A mAbs DX1 and DX12 were produced in our laboratory, and HP3G10 was purchased from MBL International. Primary cell cultures and cytotoxicity assays Venous blood was obtained from healthy volunteers, under procedures ap- proved by the Human Ethics Committees of the Australian Capital Territory Downloaded from Department of Health and Community Care and the Australian National Uni- versity (Canberra, Australia) and the University of California, San Francisco, Committee on Human Research. Polyclonal NK cells were generated and tested in cytotoxicity assays as described previously (22–24). Results and Discussion LLT1 liposomes bind human NKR-P1A http://www.jimmunol.org/ We generated fluorescent-labeled liposomes containing human LLT1 and evaluated their staining of the mouse Ba/F3 pro-B cell line and BaF/3 cells stably transduced with human NKR- ϩ FIGURE 1. Binding of LLT1 liposomes to NKR-P1A cells. A, Left, P1A (BaF/3-NKR-P1A). LLT1-liposome complexes bound Amounts of NKR-P1A on untransfected BaF/3 and NKR-P1A-transfected specifically to BaF/3-NKR-P1A, but not parental BaF/3 (Fig. BaF/3 cells (solid, anti-NKR-P1A; shaded, control). Right, LLT1 liposomes 1A). This interaction was specifically blocked by anti- stain BaF/3-NKR-P1A in the presence of isotype-matched control mAb (solid NKR-P1A mAb (dashed line), but not control Ig (solid). LLT1- line), but not in the presence of anti-NKR-P1A (dashed) (10 ␮g/ml), and do liposomes also bound to the human NK cell line YT transfected not stain untransfected BaF/3.